Passage of fluid and cells out of blood vessels is a significant contributing factor to inflammation, tissue injury, and death in a variety of circumstances. These include ischemic injury, toxic shock, allergic and immune reactions. Vascular permeability is regulated in part by cell-cell adhesions between endothelial cells.
The endothelial cell monolayer lining the vasculature forms a barrier that maintains the integrity of the blood fluid compartment, but permits passage of soluble factors and leukocytes in a regulated manner. Dysregulation of this process produces vascular leakage into underlying tissues, which accompanies the inflammation associated with pathological conditions involving edema. Edema associated with vascular permeability also occurs in ischemic injury due to the secretion of vascular endothelial growth factor (VEGF) by hypoxic tissues, which increases tissue damage in animal models of stroke and myocardial infarction. Vascular permeability is characterized by altered cell-cell contacts and the appearance of paracellular pores between adjacent cells. Integrity of the endothelial barrier is regulated in part by opposing roles of the actin cytoskeleton in which cortical F-actin stabilizes cell-cell contacts, whereas intracellular stress fibers exert tension to induce permeability.
Vascular permeability is a precisely regulated function that can contribute positively to immune responses and wound healing; however, leakage of fluid and immune cells into tissues can have serious and life-threatening consequences in a variety of diseases. Fluid accumulation in the lungs because of increased permeability of the pulmonary vasculature leading to respiratory insufficiency is a key element in acute respiratory distress syndrome. Vascular leak after stroke or myocardial infarction due to the release of VEGF by hypoxic tissues substantially increases tissue injury after these events. Vascular leak and tissue edema contribute to organ failure in sepsis.
Lung injury is a serious, often fatal, medical problem (Orfanos et al., 2004; Lionetti et al., 2005). It is usually caused by infection and can be exacerbated by mechanical ventilation to trigger leakage of fluid into the lungs, leading to respiratory insufficiency. Incidence of death in acute lung injury is in the range of 30-40% and no specific treatment is currently available.
The small GTPase Rac regulates formation and function of cell-cell adhesions in a number of systems. In epithelial and endothelial cell types, Rac is important for both the assembly of adherens and tight junctions and for their disruption during cell scattering or in response to agonists that trigger permeability. These complex effects suggest that different Rac effector pathways may differentially regulate cell-cell junctions. Precise temporal and spatial regulation of Rac and its effector pathways are likely to be critical for determining the balance between strengthening and disrupting cell-cell adhesions. However, the downstream pathways that govern these effects are poorly understood.
The p21-activated kinases (PAKs) are serine/threonine kinases activated downstream of Rac and Cdc42 that participate in multiple cellular functions, including motility, morphogenesis, and angiogenesis. GTP-bound Rac and Cdc42 bind to inactive PAK, releasing steric constraints imposed by a PAK autoinhibitory domain and permitting PAK auto-phosphorylation and activation. Numerous autophosphorylation sites have been identified that serve as markers for activated PAK. Prominent PAK downstream targets include LIM kinase, which regulates actin polymerization through its effect on cofilin, and myosin light chain (MLC). PAK2 catalyzes monophosphorylation of MLC at Ser19 to increase contractility and trigger cell retraction. However, PAK can also inhibit MLC kinase and thereby limit MLC phosphorylation and retraction. Serine 141 on PAK2 is a site within the AID sequence that is phosphorylated during activation of the kinase. Phosphorylation of this site contributes to activation by blocking interaction of the AID with the kinase domain to relieve autoinhibition. In endothelial cells, expression of catalytically active PAK1 increased MLC phosphorylation and cell contractility, whereas inhibiting PAK reduced cell contractility. Thus, in these cells, the dominant effect of PAK appears to be the promotion of contractility.
It was previously demonstrated that activation of PAK in endothelial cell-cell junctions regulates vascular permeability in response to cytokines (Stockton, J. Biol. Chem. 279:46621-46630). It did so by controlling phosphorylation of myosin light chain, which promotes cell retraction. PAK is known to regulate ERK1/2 activation (Frost, 1997, EMBO J. 16:6426-6438). Erk is also known to regulate vascular permeability (Verin, Am J Physiol Lung Cell Mol Physiol. 2000 279:L360-70; Borbiev Am J Physiol Lung Cell Mol Physiol. 2003 285:L43-54).
PAK can bind to a protein called PIX (alpha or beta isoforms), which in turn binds to another protein called GIT (isoforms 1 or 2) (reviewed in Turner, Curr Opin Cell Biol., 2001, 13:593-599.) GIT has been proposed to be a scaffold protein that enhances activation of Erk MAP kinase (Yin 2004, Mol. Cell Biol. 24:875-885). The PAK pathway seems to be involved in many events that stimulate vascular leak. GIT proteins are GTPase-activating proteins for ADP-ribosylation factor (ARF) small GTP-binding proteins.
The sequence within PAK which binds to PIXα and PIXβ is PPPVIAPRPEHTKSVYTR (SEQ ID NO:1) (Manser et al., Mol. Cell. 1998 1:2:183-92). The core sequence within PIX that binds GIT1/2 is: AALEEDAQILKVI (SEQ ID NO:2), corresponding to amino acid residues 685-698 for PIXα and amino acid residues 527-542 for PIXβ (Feng et al., 2002, J. Biol. Chem. 277:5644-5650). The core sequences within GIT1 and GIT2 that bind PIX proteins are the Spa2 homology domains (255-375 in GIT1) and the coiled coil regions (428-485 in GIT1) (Premont et al., 2004, Cell Signal 16:1001-1011). The region with GIT1 that binds MEK is also within the Spa homology domain (amino acid residues 255-375) (Haendeler et al., 2003, J. Biol. Chem. 278:50:49936-44).
PAK kinase is activated by the small GTPases Rac and Cdc42. It is sometimes found in a complex with two other proteins, namely PIX (α or β) and GIT (1 or 2). Pak binds directly to the PIX SH3 domain through an unconventional proline rich sequence in PAK. PIX then associates with GIT proteins through the extreme C-terminus of PIX and two regions within GIT: the Spa2 homology domains (amino acid residues 270-363) and the coiled coil (amino acid residues 428-485). GIT1 has also been found to be a scaffold protein for activation of Erk. GIT1 binds MEK1/2, the upstream kinases for Erk activation.
It was previously shown by Applicants that PAK kinase is critical for induction of vascular permeability by growth factors, inflammatory cytokines, and thrombin. PAK controlled permeability by regulation phosphorylation of myosin light chain kinase and increased cell contraction, which disrupts the cell-cell junctions that serve as a permeability barrier.
There is a long felt need in the art for compositions and methods to regulate vascular permeability. The present invention satisfies these needs.